Central to understanding how eukaryotic cells and the viruses that proliferate within them regulate the replication of their genomes is understanding the nature of DNA sites where replication begins and the proteins that interact with them. Previously, we and others have developed various strategies for mapping the locations of origins of bidirectional replication (OBRs) in the chromosomes of eukaryotic cells based on quantitative analyses of the amounts and distribution of nascent DNA labeled during its biosynthesis. These methods have been used by us as well as others to identify a number of replication origins in metazoan chromosomes to a resolution of #1 kilobase (kb), suggesting that metazoan chromosomes contain specific replication origins analogous to those found in the genomes of simpler organisms (e.g. yeast, protozoa). However, using 2D gel electrophoresis to detect structures of replication bubbles and forks, others have concluded that initiation events in metazoan cells occur randomly throughout initiation zones as large as 55 kb. During the past year:1) We have identified two primary initiation sites for DNA replication in the hamster DHFR gene initiation zone.2) We have shown that mammalian replication origins are not defined by their attachments to the nucleoskeleton.3) We have shown that some replication origins are highly methylated and some are not. The ones that are methylated are rapidly remethyated, and their specific activity is either directly or indirectly dependent on DNA methylation.4) We have discovered how to selectively activate primary initiation sites in mammalian nuclei using a Xenopus egg extract5) We have discovered that pre-replication complexes in mammalian nuclei are assembled de novo during G1-phase at specific chromosomal sites, and that this assembly involves the differential binding of Orc1 and Orc2 proteins to mammalian chromatin.6) We have been able to show that Orc1 and Orc2 proteins are bound to the same specific chromosomal sites that act as origins of bidirectional DNA replication.